Aqueous two-phase systems have widespread use in biochemistry and biotechnology for purifying biological materials such as cells, proteins, nucleic acids and steroids (see e.g. P.-Å. Albertsson, Partition of cell particles and macromolecules, 3rd ed., Wiley, New York City, N.Y., USA (1986) and H. Walter et al, Partitioning in Aqueous Two-Phase Systems, Academic Press, Orlando, Fla., USA (1985)). The systems are suitable for biological materials because each phase contains about 70 to 90% by weight of water, thereby substantially reducing the risk of denaturation of biomolecules such as proteins (H. Walter et al, Aqueous two-phase systems, Methods in Enzymology, vol. 228, Academic Press, San Diego, Calif., USA (1994)).
The aqueous two-phase systems are composed of two immiscible polymeric materials, one polymeric material in combination with a high salt concentration or one polymeric material in combination with a surfactant. Elevating the concentrations above a certain critical value produces two immiscible aqueous phases in which the polymeric materials, polymeric material and salt or polymeric material and surfactant are partitioned.
The partitioning of proteins in aqueous two-phase systems mainly depends upon protein hydrophobicity, charge and size. The partitioning can be influenced by changing polymeric materials, the molecular weight of the polymeric materials, the pH and by adding salts to the system (G. Johansson, Acta Chem. Scand., B 28 (1974), pp. 873–882).
Aqueous two-phase systems can be scaled up readily, since the partitioning of biological materials such as proteins is essentially independent of the size of the system. The time for phase separation can, however, be prolonged in large-scale systems depending e.g. on the geometry of the separation vessel.
On a laboratory scale, use is commonly made of dextran and polyethylene glycol (PEG) as the immiscible polymeric materials. Dextran is, however, a relatively expensive polymeric material and for large-scale purification, e.g. industrial scale enzyme extraction, various combinations of PEG and salts are more frequent (K. Köhler et al, Methods in Enzymology, vol. 228, Academic Press, Orlando, Fla., USA, (1994), pp. 627–640).
U.S. Pat. No. 4,740,304 to Perstorp AB relates to compositions containing hydroxyalkyl starch for use in systems with two or more phases for extraction, purification, concentration and/or separation of biological substances. In one preferred embodiment, the hydroxyalkyl starch is hydroxypropyl starch (HPS). In another preferred embodiment the hydroxyalkyl starch is combined with another polymer, e.g. polyethylene glycol (PEG) or polypropylene glycol. In the examples of U.S. Pat. No. 4,740,304, use is made of various enzymes.
The use of aqueous two-phase systems for purifying biomolecules has been limited, however, since the target products have been contaminated with a phase-system polymer, thus necessitating additional and complicated purification steps. Thus, hitherto the target products to be purified have been partitioned to a salt solution or remained dissolved together with a phase-system polymer. To alleviate this problem, the use of thermo-separating polymeric materials in aqueous two-phase systems has been introduced. This makes it possible to perform temperature-induced phase separation whereby the target biomolecule can be separated from the polymeric material in a very efficient way. This technique has been utilized on a laboratory scale for purifying various enzymes. Thus, temperature-induced phase separation has been used to purify 3-phosphoglycerate kinase and hexokinase from baker's yeast homogenate (P. A. Harris et al, Bioseparation, vol. 2 (1991) pp. 237–246). Furthermore, temperature-induced phase separation has been used to purify two ecdysteroids and glucose-6-phosphate dehydrogenase (P. A. Aired et al, J. Chromatogr., vol. 628 (1993) pp. 205–214 and P. A. Aired et al, Bioseparation, vol. 2 (1992), pp. 363–373, respectively). P. A. Alred et al, J. Chromatogr. A, 659 (1994) pp. 289–298, also discloses temperature-induced phase separation for purifying glucose-6-phosphate dehydrogenase, hexokinase and 3-phosphoglycerate kinase from baker's yeast.
EP-A-262651 to Union Carbide relates to a method for recovering enzymes from aqueous solutions which contains at least one polymeric material exhibiting inverse solubility characteristics. The method comprises elevating the temperature of the solution above the temperature of precipitation of the polymeric material and separating the polymeric precipitate from the enzyme-containing solution. The polymeric material is preferably selected from polyalkylene glycols, such as polyethylene or polypropylene glycol, poly(oxyalkylene) polymers or copolymers, ethoxylated surfactants, silicone-modified polyethers and polyvinyl pyrrolidone. The temperature is suitably elevated to a temperature less than about 90° C., preferably between about 50° C. and about 75° C. In the examples of EP-A-262651, use is made of λ-amylase.
EP-A-0 574 050 to Gist-Brocades relates to large-scale separation and purification of hydrophobic fermentation products. The method comprises adding to a mixture of the desired product and contaminants a non-ionic surfactant, a flocculating agent, an extra surfactant and a salt. The fermentation product is suitably a protein and preferably an enzyme, which can be used in detergent compositions.
WO 96/23061 to Genencor International relates to a surfactant-based enzyme extraction process, wherein a hydrophilic fermentation product, especially a detergent-type enzyme, is purified by contacting a clarified or whole fermentation broth containing the desired product with one or more salt(s) and a suitable surfactant with a HBL value exceeding about 12. The fermentation broth, salt and surfactant are separated into two phases, one of which is rich in surfactant and the desired product and the other rich in salt(s).
Garg et al, Biotechnol. Appl. Biochem. 20 (1994) pp. 199–215 relates to use of a temperature-induced phase-forming detergent (Triton X-114) as ligand carrier for affinity partitioning in an aqueous three-phase system. Triton X-114 was modified with Cibacron Blue to give a detergent-dye conjugate, which was used as an affinity ligand for the enzyme lactate dehydrogenase (LDH). When an excess of detergent was used, a three-phase system with a detergent-rich middle phase was formed. The detergent-dye conjugate partitioned to this detergent-rich phase. The enzyme was recovered by harvesting the detergent-rich conjugate-containing phase and subjecting it to temperature-induced phase separation.
As is evident from the above, the use of two-phase separation has been directed primarily to purification of hydrophilic macromolecules, in particular enzymes. Use of two-phase systems for purifying hydrophobic lipoproteins, is, however, known. Thus, Wiegel et al relates to partitioning of high-density lipoproteins (HDL) in two-phase systems (J. Chromatogr. B, 661 (1994) pp. 159–164). Here use is made of dextran and PEG for separating the HDL particles. The preferred enrichment of HDL particles in the dextran-rich more hydrophilic bottom: phase is attributed to hydrogen bonding between dextran and the molecules constituting the HDL particles (the apo-protein of HDL).
The main function of lipoproteins in plasma is to transport lipids, such as cholesterol and triglycerides. There are four major classes of lipoproteins: chylo-microns (CM), very low density (VLDL), low density (LDL) and high density (HDL) lipoproteins. Of these, HDL is directly involved in the removal of cholesterol from peripheral tissues, carrying it back either to the liver or to other lipoproteins, by a mechanism known as “reverse cholesterol transport” (RCT).
The “protective” role of HDL has been confirmed in a number of studies. Recent studies directed to the protective mechanism(s) of HDL have been focused on apolipoprotein A-I (ApoA-I), the major component of HDL. High plasma levels of ApoA-I are associated with a reduced risk of CHD and presence of coronary lesions.
Several methods have been proposed for purifying lipoproteins, such as ApoA and ApoE, either from plasma or produced by recombinant DNA techniques. On a laboratory scale use is commonly made of centrifugation, ion-exchange chromatography, affinity chromatography, isoelectric focusing, gel filtration and high-performance liquid chromatography (HPLC) (see Methods in Enzymology, vol. 128, Academic Press, San Diego, Calif., USA (1986)). There is, however, a need for an additional quick, sensitive and reliable method for preparation of ApoA and ApoE, especially on an industrial or pilot-plant scale.
As stated previously, the use of two-phase separation has been directed primarily to purification of hydrophilic macromolecules. Optimization of the two-phase separation technique for purifying hydrophobic or amphiphilic compounds, would increase the number of process techniques available for purifying the hydrophobic or amphiphilic compounds, e.g. lipoproteins such as ApoA or ApoE. It is the purpose of the present invention to provide such a technique.